METABOLIC ABNORMALITIES DETECTED IN A SURVEYOF MENTALLY BACKWARD INDIVIDUALS IN

NORTHERN IRELANDBY

NINA A. J. CARSON and D. W. NEILLFrom the Royal Belfast Hospital for Sick Children and the Royal Victoria Hospital, Belfast

(RECEIVED FOR PUBLICATION JUNE 8, 1962)

In 1908, Sir Archibald Garrod in his Croonianlectures coined the name 'inborn errors of meta-bolism' to describe a group of rare metabolicdisorders which appeared to be genetically deter-mined. In 1923, he described six such conditions ina monograph-alcaptonuria, pentosuria, cystinuria,albinism, pancreatic steatorrhoea and porphyria.He predicted that a biochemical reason would befound for these disorders and that they probablyresulted from a block at some point in metabolismdue to the congenital deficiency of a specific enzyme.The development of chromatographic and enzymatictechniques over the past 15 years has led to theelucidation of many metabolic pathways and pro-vided the scientific proof of his brilliant induction.Garrod thought that in alcaptonuria, the accumu-

lation of homogentisic acid was evidence that thissubstance was a normal metabolite in the breakdownof tyrosine and that its increase was due to a failureof oxidation of homogentisic acid. Now, a halfcentury later, Garrod's hypothesis has been provedby demonstration of the absence of homogentisicacid oxidase activity in the liver of a patient withalcaptonuria (La Du, Zannoni, Laster and Seeg-miller, 1958).

In the past decade many abnormalities in theexcretion of aminQ acids have been noted in asso-ciation with hereditary biochemical disorders andmental retardation. Among these may be notedmaple syrup urine disease (Dancis, Levitz, Millerand Westall, 1959), in which there is a metabolicblock in the degradative pathway of the branchedchain amino acids isoleucine, leucine and valine;this is associated with mental retardation, muscularhypertonicity and a maple syrup odour to the urine.Hartnup disease was first described by Baron, Dent,Harris, Hart and Jepson in 1956; this is character-ized by a pellagra-like, light-sensitive skin rash,cerebellar ataxia and emotional instability. Aconstant feature of the disease is a generalizedaminoaciduria related to an intermittent upset in

tryptophane metabolism (Milne, Crawford, Giraoand Loughridge, 1960). Harris, Penrose andThomas (1959) described cystathionuria in an elderlymentally-retarded patient. Glycinuria has recentlybeen described by Childs, Nyhan, Borden, Bard andCooke (1961) where mental retardation and micro-cephaly were accompanied by excess glycine in urineand blood.

Metabolic errors in carbohydrate metabolismhave been known for many years; pentosuria ascited by Garrod in 1908 was first described in 1892by Salkowski and Jastrowitz. Galactosaemia wasalso first noted in 1908 by Reuss-although themechanism of its occurrence was only described in1956 by Schwarz, Golberg, Komrower and Holzelin England and Isselbacher, Anderson, Kurahashiand Kalckar in America. Gargoylism was recog-nized in 1917 by Hunter, but it is only within recentyears that it has been discovered to be due to devia-tions in the metabolism of mucopolysaccharides(Dorfman and Lorincz, 1957; Meyer, Hoffman andLinker, 1957).

All these conditions are rare and there are, as yet,few specific instances in which a chemical distur-bance results in mental retardation; less than5% of the mentally defective population are of thistype. With the exception of Hartnup disease, thesemetabolic disturbances are responsible for the moresevere degrees of mental retardation. Some areamenable to treatment especially when diagnosedand treated from an early age, e.g. phenylketonuria,galactosaemia, maple syrup urine, but in them theactual cause of the brain damage is still unknown.With the increased interest in these metabolic

errors in recent years it was considered desirable todetermine their incidence in Northern Ireland.With special reference to phenylketonuria it wasfelt that the registration of all phenylketonuricfamilies would allow the condition to be anticipatedin any new siblings so that treatment could be startedat an early age when success would be more sure.

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There are approximately 4,000 mentally retardedpersons in Northern Ireland who are registered asbeing under the supervision of the Special Carebranch of the Northern Ireland Hospitals Authority.This includes those persons, irrespective of age orsex, who have an intelligence quotient below 75,indicating mental retardation. Of all cases reportedto the Special Care Service, 25% are high gradedefectives with an I.Q. varying between 50-75,approximately 60% are medium grade with anI.Q. between 30-50, and 15°, are low grade.Of these people, 5000 are cared for in the com-

munity under the supervision of medical officers andsocial workers of the Special Care Service, theremaining 50% being cared for in residential insti-tutions for the mentally retarded.

This paper is an interim report of studies ofbiochemical disturbances which cause brain damage,mainly as a result of some inborn error of meta-bolism. Samples of urine were obtained from thepatients and every urine was subjected to a battery,of simple qualitative tests in order to exclude suchobvious abnormalities as excess reducing substances,proteinuria, phenylpyruvic aciduria, excess indican-,uria, sulphur-containing amino acids, ketones,porphobilinogen, mucopolysaccharides, or abnor-malities of calcium excretion.

Irrespective of the findings of these qualitativetests, the urinary excretion of amino acids usingpaper chromatographic techniques was studied.Urine was used for these tests as it is the most easilyobtainable biological fluid from mentally retardedpatients as well as being a rich source of metabolites.In the event of an aminoaciduria or other abnor-mality being detected, fresh samples of urine wereobtained in order to confirm the finding beforefurther studies were undertaken.

Material and MethodsAs it was impossible in a survey of this kind to obtain

absolutely fresh specimens, thymol crystals were addedto the sample bottles before the urine was collected;on receipt of the urine it was stored in a refrigeratoruntil the tests were performed.

A. Qualitative Screening TestsReducing substances were detected by the use of Ames

Clinitest tablets. In the event of a positive reaction theurine was examined by one-dimensional chromatographyto identify the carbohydrate, if present.

Phenylpyruvic acid was detected by the use of eitherfilter paper strips impregnated with ferric chloride andbuffered (Ames Phenistix), or by the ferric chloride test.

Ketones and ketoacids were detected by using theRothera nitroprusside test in the form of the com-mercially available Acetest tablets, and by the additionof 2-4 dinitrophenylhydrazine solution with formationof the characteristic dinitrophenylhydrazones.

Protein was detected by addition of sulphosalicylic acid.Sulphur-containing amino acids were detected by the

sodium cyanide-nitroprusside test as described byBrand, Harris and Biloon (1930).

Porphobilinogen was detected using the Watson modi-fication of Ehrlich's reagent, with partition in amylalcohol/benzyl alcohol to distinguish porphobilinogenfrom urobilinogen.

Gross errors in bone metabolism were excluded by use ofSulkowitch qualitative reagent for calcium.

Indican (potassium indoxyl sulphate) was detected byObermeyer's Prussian blue technique.

Urinary indican usually arises from indole formed asa result of bacterial action on dietary tryptophane in theintestine, the indole is then absorbed and transported tothe liver where it is oxidized to indoxyl and conjugatedto sulphate. This indican is then excreted in the urine.Urinary indican, according to Sprince (1961), is increasedin many conditions such as renal insufficiency, achlor-hydria, certain anaemias, intestinal ulceration, peritonitis,systemic infections and bowel stasis. Increased levels ofurinary indican have also been described in Hartnupdisease (Milne et al., 1960) and in phenylketonuria(Bessman and Tada, 1960). It has been suggested byC. E. Dent (personal communication) that those urineswhich gave a strongly positive reaction for indicanmight possibly have some abnormality of indole meta-bolism, and indicanuria was used as a rough screeningtest to select those urines in which further investigationfor indole derivatives might prove informative.

Excess acid mucopolysaccharides: The specific stainingreaction with Alcian blue was used to detect the increasein acid mucopolysaccharides found in gargoylism. Amodification of the technique described by Heremans andVaerman (1958) was adopted: 10 yd. urine was appliedto Whatman No. 4 filter paper, dried, dipped in a solu-tion of 1% Alcian blue dissolved in 90% glacial aceticacid and the background colour washed out with waterfollowed by glacial acetic acid and finally water. Acidmucopolysaccharides stain bright blue against a whitebackground.

B. Amino Acid Chromatography. All urines wereexamined for abnormality of amino acid excretion. The'rack' tank of Datta, Dent and Harris (1950) was usedwith certain routine modifications to make their inter-pretation semi-quantitative. Total nitrogen estimationswere performed on each urine and the volume ofurine containing 250 ,ug. nitrogen was applied toeach paper. Whatman's No. 1 chromatography paperwas used. Each urine was desalted by electrodialysisusing ion exchange membranes (Smith's modification ofWood's method, 1960) before spotting it onto the paper.The solvent system used was butanol-pyridine-water1: 1: 1 as a first solvent, followed by phenol ammoniaas a second solvent (Smith, 1960).The amino acids on the developed chromatogram

were visualized by dipping in a 0.1% solution of nin-hydrin in acetone; they were then dried at room tem-perature for about 30 minutes and heated at 800 C. forfive to seven minutes.

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METABOLIC ABNORMALITIESIn order to make the results semi-quantitative 51A1.

of x-amino octanoic acid was applied to each paperand the density of this spot was taken to represent 5.The density of other spots was numbered from I to 10with reference to this spot. This permitted the amountof each amino acid excreted to be recorded as a figure.In view of the subjective nature of this assessment allthe sheets were reviewed by one person.To establish the normal range of values for the urinary

excretion of each amino acid as detected by the abovechromatographic techniques, samples from the com-munity were picked in the following way:

All children who attended State-aided schools (980oof population) in Belfast County Borough (i.e. approxi-mately 70,000 children) and who were listed in the recordsof the schools medical service as being born on January Iand on July 1 from 1946 to 1955 inclusive were admittedas controls, i.e. children between the ages of 5 and 15years.

It was thought prudent to choose the dates shown toexclude the possibility of a seasonal incidence of acongenital anomaly. The age-group was chosen asbeing the only practicable group in which a represen-tative sample of the population with normal intelligencecould be drawn and examined. Although other workersin this field claim that there is no change in the aminoacid excretory pattern with age, after the first two yearsof life (Bigwood, Crokaert, Schram, Soupart and Vis,1959), it was considered advisable to examine a furthersample of urines from normal adults. To do this weexamined 100 samples from age-group 18-50 years;these consisted mainly of healthy nursing, medical andtechnical personnel of hospitals. No significant differ-ence was found in the two age-groups.The principal amino acids normally excreted are

glycine, serine, alanine. glutamine, glutamic acid,histidine, taurine and smaller amounts of lysine, cystine,leucine, tyTosine, threonine, aspartic acid and occasion-ally methyl histidine. The upper and lower limits ofexcretion of these amino acids were determined in theabove control group, a range of normality thus beingestablished for the chromatographic techniques used.

ResultsThe vast majority of urines from mentally

retarded persons showed an excretion of aminoacids within the normal range: there were no par-ticular patterns or trends characteristic of 'mentalretardation', a fact found by other workers in thisfield. With a few exceptions, all the abnormalitiesdiscovered were obvious departures from the normaland left no room for doubt. The abnormalitiesdetected in this way can be listed.

1.

.3.4.5.

6.

Phenylketonuria.Argininosuccinic aciduria.Homocystinuria.Miscellaneous aminoacidurias.Cerebral-ocular-renal syndrome of Lowe.Galactosaemia.

7. Lignac-Fanconi type syndrome with mentalretardation.

8. Gargoylism.The further investigations carried out can be

considered under these headings.

Phenylketonuria. This was one of the firstinborn errors of metabolism to be discovered sinceGarrod's work on cystinuria and alcaptonuria, andis due to a rare recessive gene in homozygous form(Penrose, 1935). The abnormality was first dis-covered by Foiling (1934) who, when testing urinesof mentally retarded people with dilute ferricchloride, found a green colour appearing in a fewurines which faded on standing. This reaction wasdue to the large quantities of phenylpyruvic acid inthe urine of these people. Jervis. Block, Bollingand Kanze (1940) established that the excretion ofthis substance was not the direct result of the inbornerror of metabolism but that it was an abnormalmetabolite from the breakdown of the amino acidphenylalanine which, due to the deficiency or lackof its specific enzyme, phenylalanine hydroxylase,could not be converted to tyrosine. but built up tohigh concentrations in the body and the phenyl-pyruvic acid production resulted from metabolismof phenylalanine by the main alternative pathwav.The diagnosis of phenylketonuria depends on the

following tests:i. 1000 ferric chloride or 'phenistix .

ii. Dinitrophenylhydrazine test.iii. Presence of phenylalanine in the amino acid

chromatogram.Test i. may become negatixve after a period of

a few days if urine is not kept cold and free frombacterial contamination, and a negative ferricchloride on an old urine is of no -alue. The finaldiagnosis is made by chromatography.The chromatographic solvent sy-stem used (Smith,

1960) results in phenylalanine. leucine and iso-leucine being superimposed on one another. Inorder to make sure that the spot was phenylalanine aone-dimensional chromatogram was run on a stripof Whatman No. 4 paper 22 in. long using a watersaturated amyl alcohol solvent in an atmosphere ofdiethylamine and allow ing it to run for approxi-mately 48 hours when isoleucine. leucine andphenylalanine can all be separated out as threediscrete spots. This was performed in all casesthought to be phenylketonuria.On screening 2,081 urines, 49 cases of phenyl-

ketonuria were discovered in 36 families. 11 famnilieshaving two or more siblings suffering from thedisorder. Their ages ranged from 7 days to 59years; 27 were males and 22 were females. Of the

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2,081 urines tested, 81 were from mentally deficientchildren directly referred from paediatric consul-tants and, of these, nine were phenylketonurics.If these figures are statistically treated so as toinclude only those individuals under Special Care,then the incidence is seen to be 2%.The majority of these cases had irreparable brain

damage and were diagnosed too late to benefitfrom treatment. Five patients, however, receiveddietary treatment.

Case 1. F.J.G. was diagnosed at age 7 days; thephenistix' test was still negative, the dinitrophenyl-hydrazine test negative, but chromatography revealeda large spot of phenylalanine. This child was placedon a diet at 2 weeks of age and is making satisfactoryprogress at the time of writing (age 8 months). Thisinfant was tested at this early stage as an older siblingwas known to be suffering from phenylketonuria.

Case 2. A.D. was diagnosed at 3 weeks of age bythe health visitor during a routine visit, using phenistix;this was confirmed by chromatography when largeamounts of phenylalanine were found in the urine andblood.

Case 3. E.G. was diagnosed at 24 years, her parentshaving brought her to hospital owing to retarded develop-ment (she did not sit up unaided until age of 18 months)and had minor convulsions. She was placed on a dietand is now aged 7 years with an I.Q. of 87.

Case 4. E.N. Although this child had a brotherwho was known to be suffering from phenylketonuriashe did not come to the attention of the paediatricianuntil she was 8 months old. She did not appear to bevery retarded and she was immediately put on to thespecial diet. Dietary management has been difficult inthis case as the child showed marked dislike for the tasteof her special food and a period of tube-feeding for sixmonths was required after which oral feeding wasrecommenced successfully. She is now aged 34 yearsand although formal testing was not possible thepsychologist reports 'good enough normal behaviour andability for her age'.

Case 5. G.McM. was diagnosed at 10 days. Therewere two other mentally retarded siblings in the family,both suffering from phenylketonuria. The child is nowaged 7 years, is progressing satisfactorily and is attendingordinary school. She still remains on her phenyl-alanine-restricted diet.

Argininosuccinic Aciduria. This was first des-cribed in 1958 by Allan, Cusworth, Dent and Wilson.The clinical findings are mental retardation, non-

progressive ataxia with abnormal electroencephalo-gram pattern which may be associated with epilepticfits, abnormal friable hair, possible systolic murmurs,and excretion in the urine of argininosuccinic acidin large quantities.

Two cases of argininosuccinic acid excretors werediscovered from one sibship. They are at presentbeing investigated; both were very slow to walk andone has had intensive investigation carried out a fewyears ago because of ataxia. They were mentallyretarded and large amounts of argininosuccinicacid were found in their urine. Fig. 1 a shows achromatogram with a large spot of argininosuccinicacid.

Homocystinuria (2 cases).* This appears to bea metabolic error, previously undescribed, occurringin two female siblings, aged 5 and 7 years. Thereis one other normal male sibling aged 2 years.The findings were similar in the two cases with a fewexceptions.

Case Reports. Both girls had a history of fits, theirdevelopmental milestones were slow and they were bothseverely mentally retarded. On physical examinationthe similarity in their appearance was very striking.They were dull looking, obviously mentally retardedchildren with fair hair, very red cheeks and knock knees.Their hair was abnormal in that it grew very slowly,and especially round the occiput where it broke off afteronly a few inches of growth. There was a peculiarmottling of the skin similar to ephelis ab igne and this wasespecially noticeable on lower limbs. The skin tendedto be rather dry especially on the extensor surfaces ofthe arms. Both children had dislocated lenses, causingmarked iridodonesis on movement of the pupils. Therewas nothing abnormal in the respiratory or cardio-vascular systems. The older child had a protuberantabdomen with hepatomegaly.The following investigations were carried out:A radiograph of the skeleton was normal; audiometry

tests were normal; E.E.G. normal; E.C.G. normal;I.Q. 30.

Liver biopsy in older child showed fatty degeneration.Biochemistry tests were normal, except for two liver

enzymes, isocitricdehydrogenase and leucine amino-peptidase, both of which were raised on two occasions.

Haematological investigation revealed a moderatemacrocytic normochromic anaemia more pronounced inthe child with hepatomegaly.

Routine screening tests revealed a positive sodiumnitro-prusside-cyanide test indicating an increasedexcretion of a sulphur-containing amino acid. Aminoacid chromatography at this stage showed a normalchromatogram but on oxidizing the urine after applica-tion to the filter paper with peroxide and ammoniummolybdate the chromatogram showed what appearedto be an increase in cysteic acid with an otherwise normalpicture (Fig. lb). On repeating the chromatogram withadded L-cystine and oxidizing, only one spot in thecysteic acid position was seen.

* Since this paper was written a further five cases of homo-cystinuria have been found in three sibships. These will be thesubject of a further report.

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METABOLIC ABNORMALITIESAi'

FIG. la.-Argininosuccinic acid.

The only conditions so far known where there is amarked increase in cystine excretion are: (1) cystinuria-here the overexcretion of cystine is accompanied by anincrease in the excretion of the basic amino acids,lysine, ornithine and arginine; and (2) Wilson's diseaseand Lignac-Fanconi disease where the cystinuria is partof a marked general aminoaciduria.

The older child presented with hepatomegaly, andexcess cystine excretion in mild liver disease has beendescribed (Dent and Walshe, 1951). It was however feltthat this finding in two siblings showing similar clinicalfeatures merited further study and, accordinglyspecimens of the urines were sent to Professor Dent'sgroup at the University College Hospital MedicalSchool, London, who qualitatively confirmed theoverexcretion of cystine. However, on submitting24-hour specimens to Professor Dent for quantitativestudy it was discovered that the amino acid involved wasnot cystine but homocystine. At the same time thechromatograms were repeated here, having given one ofthe children a loading dose of L-cystine, and in theoxidized papers it was discovered that there were in facttwo spots side by side in the cysteic acid position, one

of these being homocysteic acid. Further metabolicstudies are being undertaken at present in these twochildren in association with University College Hospital,London.

Miscellaneous Aminoacidurias. In the 2,081 urinesexamined there were two cases of very severe

generalized aminoaciduria and 36 cases of moderateseverity; these latter are of 'central cluster' patterninvolving glycine, serine, alanine, glutamine and/orglutamic acid, histidine, threonine and sometimestaurine.

FIG. Ib.-Homocysteic acid.

There is a further miscellaneous group of specificaminoacidurias, e.g. excess glycine with otheramino acids being present in normal quantities;this has recently been described in association withmental retardation by Childs et al. (1961). Alani-nuria and histidinuria were also observed. Thislatter may be associated with dietary factors, sinceit is known that histidinuria may be present aftera heavy protein meal especially in children; it is alsoincreased during pregnancy (Soupart, 1954) and atthe beginning of the luteal phase of the menstrualcycle (Soupart, 1960). However, Ghadimi, Parting-ton and Hunter (1961) have described a familialdisturbance of histidine metabolism in two sisters,one of whom had markedly retarded speech.

In three of the 444 normal control urines and ineight of the 2,081 urines from mentally retardedpatients, a pattern suggesting cystinuria wasobserved on the amino acid chromatograms. Thisconsisted of excess cystine accompanied by excessof one or more of the basic amino acids, lysine,ornithine and arginine.

It is appreciated that many of the above amino-acidurias may have a metabolic basis quite unrelatedto mental retardation, and it is hoped to study thesecases in detail.

Cerebral-ocular-renal Syndrome of Lowe. Thissyndrome was described in 1952 by Lowe, Terreyand MacLachlan from Boston. The main findingsin the syndrome are mental retardation, acidosis,eye changes (hydrophthalmos, glaucoma and /or

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..relal

FIG. lc.Ocular-cerebral-renal syndrome of Lowe.

cataracts), aminoaciduria, albuminuria (no glyco-suria), reduced ability of the kidneys to formammonia, and in the untreated cases, rickets andosteomalacia.

Case Report. One case of this syndrome was dis-covered in a male child aged 4 years, who first came tothe notice of the paediatrician with what was thoughtto be nephritis. The child was mentally retarded andbilateral cataracts were present which were first noticedat the age of 3 days and have been needled on threeoccasions. The other symptoms present were as des-cribed above. There was a severe kidney lesion withalbuminuria, gross general aminoaciduria (no glycosuria),reduced ability of kidneys to form ammonia, rickets andthe serum alkali reserve always in the acidotic range.This patient is the fourth and only surviving child. Thefirst sibling died a few hours after birth from exposureas no one was with the mother at the birth; the seconddied at 9 days with haemorrhagic diathesis and con-vulsions. The third sibling had cataracts which werenoticed during the first few days of life. He was not arobust baby and soon after an operation for extraction ofcarious teeth at the age of 3 years the child died, hiscondition undiagnosed.

Fig. lc shows the aminoaciduria found in this casecompared with that of a normal pattern. The chromato-gram shows a general aminoaciduria with special pre-dominance of alanine, glutamine and serine; the'nephrosis peptide' (Dent, 1948) was also present.On the question of treatment, all one can hope to do

is to keep the bony changes in check by a large intakeof vitamin D and alkalis. This has caused arrest ofthe rickets in this child, but there has been no improve-ment in his mental state or cataracts.

Galactosaemia. This is an inborn error in carbo-hydrate metabolism, first described in 1908 byReuss.

Case 1. S.C. aged 3 months. Referred to hospitalbecause of failure to thrive associated with poor feedingand infrequent vomiting. On examination, the infantwas grossly underweight being only 8 oz. (0.23 kg.) overhis birth weight. His liver was palpable two finger-breadths below the costal margin, the spleen was justpalpable, and bilateral cataracts were present. Areducing substance was present in the urine by Benedict'stest and this was found to be galactose on examinationby paper chromatography. The infant was placed ona lactose-free diet, as a result of which the liver decreasedin size and the cataracts disappeared. He was dischargedat the age of 9 months, gaining weight, but his mentaldevelopment was only that of a 4-month-old infant. At20 months he was reviewed and appeared to be aphysically normal child with a mental age of 15 months.He was still on his diet and his urine showed no reducingsubstances.

Case 2. J.C., brother of S.C., aged 9 years. Thischild came to our attention only after his youngerbrother had been diagnosed as suffering from galacto-saemia. He was mentally retarded with a mental age of5 years. At birth he was a slow feeder but apart fromthis there was little else of note in the early history.Cataracts of both eyes were present from just under1 year, and his developmental milestones were slow.He sat up at 10 months, talked at 4 years and had poorsphincter control. On the first occasion his urine wasexamined no galactose was found, but on repeating theexamination at a later date he was found to be excretinggalactose. On the performance of a modified galactosetolerance test he was found to handle the galactose in anabnormal manner. There were four other normalsiblings in the family.

Case 3. N.C., first seen at 4 weeks with jaundiceand umbilical sepsis; she was treated with antibiotics.When next seen at age 3 months she was pale and listlessand had diarrhoea and frequent vomiting. On investiga-tion she was found to be excreting a reducing substancein her urine, which proved to be galactose. The childis now normal physically and mentally and is attendingordinary school; she is still on a lactose-free diet.

This error in metabolism is inherited as an auto-somal recessive, and heterozygotes of the trait maybe detected by estimation of their galactose-l-puridyl transferase activity, the level of which liesbetween the extremely low levels of the galacto-saemic and that of a normal individual. The casesrecorded here were diagnosed by finding a reducingsubstance in the urine, which was proved bychromatography to be galactose (Fig. Id).

Lignac-Fanconi Type Syndrome Associated withMental Retardation. The following case reportillustrates the condition.

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METABOLIC ABNORMALITIESCase Report. Boy, A.S., aged 7 years. Only child

of unrelated parents. In the 12 months before his deaththis child showed many of the symptoms and biochemis-try associated with Lignac-Fanconi disease (Bickel,Smallwood, Smellie and Hickmans, 1952). Renaltubular damage was evident as seen by polyuria andexcessive thirst, albuminuria, intermittent mild glyco-suria, aminoaciduria and hypopotassaemia. Ureawas normal. Sodium cyanide/nitroprusside test wasstrongly positive, indicating the excretion of a largeamount of sulphur-containing amino acid. A chromato-gram of the urine showed a moderately severe generalizedaminoaciduria of mainly central pattern, cystine beingpresent in large quantities. Radiographs showed ageneralized decalcification of the skeleton. This wasassociated with low to normal calcium, normal phos-phorus and alkaline phosphatase, and a persistently lowalkali reserve. Steatorrhoea was present. Cataractsand dislocation of lenses of both eyes were present. Nocystine crystals were noted under slit-lamp examination.The child was mentally retarded.Hepatomegaly was present associated with liver

dysfunction as seen by abnormal results of brom-sulphalein dye test, iso-citric-dehydrogenase and theprothrombin. Liver biopsy showed the presence offatty degeneration; this has been described by Bickelet al. (1952), and one interesting case associated withcirrhosis of liver was reported by Baber (1956). Theunusual finding in this case was cyanosis of the face andextremities, which was continuously observed; this wasaccompanied in the last year of his life by severe throm-botic attacks of first one limb and then another; thesenecessitated admission to hospital and treatment withantibiotics and anticoagulants. No cardiac lesion couldbe detected. There was generalized wasting and poormuscle tone and walking became progressively difficult.The child unfortunately died at home, aged 8 years,

the parents refusing any more admissions to hospital,and no autopsy was performed.There was no family history of any mental retardation

or of the above disease. Mental retardation has notbeen described in association with the Lignac-Fanconidisease and may well represent an incidental finding.The amino acid chromatogram showed a generalized

aminoaciduria with an especially marked cystine excre-tion. Serum chromatogram of amino acids in this caseshowed a normal pattern, the aminoaciduria thereforebeing of renal origin.

Gargoylism or Hurler's Syndrome. This is ametabolic error involving polysaccharide metabolism(Hurler, 1919).

Case 1. G.G. shows all the clinical manifestationsof the syndrome. Diagnosed at 7 months by typicalskeletal deformities and radiographs. Screening testswere positive. Severe mental retardation is now presentat age 3 years.

Case 2. A.McM. Referred to the paediatric con-sultant at the age of 3 weeks with a congenital heart

FIG. ld.-(a) Patient's urine. (c) Normal urine + glucose.(b) Patient's urine + galacto6e. (d) Galactose + glucose.

lesion. She was next seen at the age of 14 months whentypical features of gargoylism were apparent and mentalretardation was evident. Screening tests were positive.

Case 3. D.P. first came to the attention of the paedia-trician at the age of 17 months because of repeated upperrespiratory infections and progressive deafness whichwas first apparent at the age of 6 months. Theappearance of the child was typical of the above con-dition; hepatosplenomegaly was present, radiographsshowed typical joint deformities, some opacity of corneawas present and the child appeared to be backward.Screening tests were positive.

Discussion

The description by Folling (1934) of oligophreniaphenylpyruvica and the elucidation of the enzymedefect involved amply confirmed the earlier work ofGarrod on metabolic errors and gave a powerfulstimulus to the search for metabolic bases of mentaldeficiency. In this context the metabolism of aminoacids as revealed by abnormalities of urinaryexcretion has been a fruitful field of research andvarious metabolic errors have been detected.The technique of multiple chromatography on

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paper makes it feasible to examine large numbers ofurine samples for abnormalities of amino acidexcretion, although it is important to appreciate thatonly relatively gross abnormalities can be detectedby a qualitative procedure of this type.The aminoacidurias can be the result of two

different mechanisms (Dent, 1947). In the 'over-flow' type, some change in the metabolism or in thedietary intake results in an increased concentrationof one or more amino acids in the blood. Renaltubular reabsorption is inadequate to conserve theexcess amino acids to the body and aminoaciduriaresults. Raised serum and urinary amino nitrogenconcentrations thus characterize this urinary excre-tion pattern as in phenylketonuria in which phenyl-alanine occurs in high concentrations in both urineand blood and indeed in the cerebrospinal fluid.The second mechanism operates in the 'renal'

aminoacidurias, in which, because of congenitaldeformity (Clay, Darmady and Hawkins, 1953),defective proximal tubular reabsorption, or toxicdamage to the tubular reabsorptive mechanism,amino acids in increased concentration appear in theurine although the blood level is normal or even low.This mechanism underlies the aminoaciduria ofgalactosaemia in which reabsorption in the proximalrenal tubule is defective. The characteristic patternof amino acid excretion in Lignac-Fanconi diseasehas been ascribed to a congenital renal deformity.The effects of toxicity can be more complex. Therenal lesions resulting from the copper abnormalityin Wilson's disease combine with the hepatic changesto produce an aminoaciduria which again is charac-teristic of the condition.

In the present study, as might be expected,metabolic disorders involving aminoacidurias ofboth types have been found. Overflow amino-aciduria is present in phenylketonuria and arginino-succinic aciduria. In the cerebro-oculo-renal syn-drome of Lowe and in Lignac-Fanconi disease theaminoaciduria is renal in origin.Of the clearly definable clinical entities found in

the 2,081 patients examined the most common hasbeen phenylketonuria with an incidence of 2%.Jervis (1953) lists the incidence of phenylketonuriaas reported from different countries and our figureof 20/ is high compared to those presented, with theexception of the figure of 2-7°, reported by Cowie(1951). The series on which this study was basedhowever was small and the figure may thus beunrepresentative. As only 500' of Special Carepatients have been examined, and until the studyis complete, it would be fruitless to speculate on thereasons for this figure of incidence. It has, however,been possible'to make use of the results so far

obtained as a guide to the initiation of early treat-ment in newborn sibs.The other less common conditions which have

been detected on the basis of the aminoacidurias towhich they give rise are being investigated further,and their discovery in this series is at once a justi-fication for the study and a stimulus to continue it.

In employing aminoaciduria as an indicator ofmetabolic disorder, it is of course important toappreciate that it is not itself the disorder, that itmay represent a secondary and minor feature of theabnormality of metabolism, as in galactosaemia,and that many such abnormalities do not give riseto an aminoaciduria. The suggestion by H.Sobotka (personal communication) that formesfrustes of gargoylism may occur undiagnosed led tothe inclusion of a simple screening test for excessconcentration of urinary mucopolysaccharide in thegroup of tests carried out on all the urine samplesin this series. The difficulties of the clinical diag-nosis suggest that much work remains to be doneto devise simple screening tests sensitive enough todetect formes frustes and heterozygous forms ofmetabolic disorders giving rise to mental defects.

SummaryUrines from 2,081 mentally retarded individuals

in Northern Ireland have been examined by quali-tative chemical tests and chromatographic techniquesin order to detect any inborn errors of metabolismwhich might be a contributory factor responsiblefor the brain damage in these people.The following abnormalities were discovered:

phenylketonuria, 49 cases; argininosuccinic aciduria,two cases; cerebro-ocular-renal syndrome of Lowe,one case; galactosaemia, three cases; gargoylism,three cases; Lignac-Fanconi syndrome, one case,a so far undescribed abnormality in cystine meta-bolism, homocystinuria, and a miscellaneous collec-tion of general and specific aminoacidurias whichare at present under investigation.

This work has been made possible by a research grantfrom the Royal Belfast Hospital for Sick Children.We wish to thank Professor F. M. B. Allen, Professor

of Child Health, Queen's University, Belfast, who wasthe instigator of this research, and the many consultantswho referred cases to us, especially Dr. Joan B. T. Logan,Dr. W. A. B. Campbell and Dr. Claude Field. Specialthanks are due to Dr. T. W. H. Weir, Dr. B. P. Glancy,Dr. V. O'Sullivan and Dr. W. R. Moffatt, consultantsin charge of Special Care Divisions, and all the manynursing staff and social workers who have taken suchtrouble to collect samples of urine for this work.We are especially indebted to Professor C. E. Dent

and his group at University College Hospital for theircontinued interest, guidance and practical help with themany problems that have arisen in this study.

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